Few processes are as fundamental to biological systems as the faithful duplication of the genetic material. In this proposal, we present our plan for continued investigation of how vaccinia virus coordinates the duplication and encapsidation of its genome. Vaccinia, the prototypic poxvirus, replicates in the cytoplasm of infected cells and possesses a high degree of autonomy from the host. Among the approximately 200 viral genes are those encoding most, if not all, of the repertoire of proteins required for DNA replication. The experimental tractability of this system, amenable to genetic, biochemical, and cell biological dissection, has made vaccinia virus an intriguing model system. The need to gain a deeper understanding of viral replication has become even more compelling in light of a growing fear that life-threatening poxvirus infections might again threaten the human population through acts of bioterrorism. Our plans for the next five years of study are: AIM I: Understanding the mechanisms of genome replication and encapsidation. Our first goal for this aim is to gain an appreciation of the strategies used by the virus to ensure specific and efficient replication of the viral genome within the cytoplasm of infected cells. First, we will explore a new hypothesis that poxviral DNA replication occurs in localized cytoplasmic domains by virtue of protein interactions with intracellular membranes. Second, we will refine our understanding of which cis-acting elements within the terminal 200-bp of the genome are important for efficient DNA replication. Our second goal is to understand the mechanism whereby progeny genomes are encapsidated into nascent virions. We will test a new model which proposes that the interaction of the telomere-bound I6 protein with the virion-bound A32 protein, a putative ATPase, mediates genome packaging. AIM II: Understanding the enzymology of genome replication. Genetic and biochemical analyses have identified a repertoire of proteins involved in vaccinia DNA replication. Our goal for these studies is to understand the assembly of a replication fork complex that can duplicate the genome in a processive manner with high fidelity. The E9 DNA polymerase, A20 processivity factor, D4 uracil DNA glycosylase, D5 NTPase, I3 single-strand DNA binding protein, and B1 protein kinase will be the major topics of study.